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Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex.

Brandon Malone1, James Chen1, Qi Wang2

  • 1Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065.

Proceedings of the National Academy of Sciences of the United States of America
|April 22, 2021
PubMed
Summary
This summary is machine-generated.

Backtracking, the reverse motion of RNA-dependent RNA polymerase (RdRp), is vital for SARS-CoV-2 transcription and replication. This process, aided by the nsp13 helicase, may enhance viral proofreading and antiviral resistance.

Keywords:
RNA-dependent RNA polymerasebacktrackingcoronaviruscryo-electron microscopymolecular dynamics

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Area of Science:

  • Virology
  • Molecular Biology
  • Structural Biology

Background:

  • Backtracking is a regulatory mechanism in cellular transcription.
  • The role of backtracking in viral transcription and replication remains largely unestablished.
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an RNA-dependent RNA polymerase (RdRp) for its lifecycle.

Purpose of the Study:

  • To investigate the role and mechanism of backtracking in SARS-CoV-2 RNA-dependent RNA polymerase (RdRp).
  • To determine the involvement of the nsp13 helicase in SARS-CoV-2 RdRp backtracking.
  • To explore the potential implications of backtracking in viral transcription, replication, and antiviral resistance.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) to visualize SARS-CoV-2 RdRp-nsp13-RNA complexes.
  • RNA-protein cross-linking to identify interaction sites.
  • Unbiased molecular dynamics (MD) simulations to characterize the backtracking process.

Main Results:

  • Structural and simulation data reveal that backtracking extrudes product RNA through the RdRp NTP entry tunnel.
  • Mismatched nucleotides at the 3' end of product RNA initiate backtracking by entering the NTP tunnel.
  • The nsp13 helicase was confirmed to stimulate SARS-CoV-2 RdRp backtracking.

Conclusions:

  • Backtracking is a significant mechanism in SARS-CoV-2 viral transcription and replication.
  • The nsp13 helicase plays a crucial role in facilitating RdRp backtracking.
  • SARS-CoV-2 RdRp backtracking may contribute to viral proofreading, impacting antiviral resistance.